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Method for immunologically measuring soluble LR11

一种免疫学测定、可溶性的技术，应用在可溶性LR11的免疫学测定领域，能够解决无法准确定量可溶性LR11、复杂、操作多等问题

Active Publication Date: 2014-01-08
SEKISUI MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As a method for measuring soluble LR11, it is known that an insoluble carrier obtained by binding a molecular chaperone RAP (receptor associated protein) having an affinity for LR11 is used to obtain a sample from a sample SDS-PAGE, Western Blot, and immunostaining with an anti-LR11 antibody after isolating soluble LR11 from a test sample (Non-Patent Documents 5 and 6), however, the process of isolating soluble LR11 from a sample is typical , the operation is many and complicated, and it is not a practical method to apply to clinical examination and other situations
[0004] The present inventors tried to establish an immunological measurement method using an anti-soluble LR11 antibody as a simple and practical method for measuring soluble LR11. When a biological source sample is used as a sample, it is known that the soluble LR11 cannot be accurately quantified due to the influence of uncertain measurement interfering substances (hereinafter, simply referred to as "interfering substances") existing in the above-mentioned samples.

Method used

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  • Method for immunologically measuring soluble LR11
  • Method for immunologically measuring soluble LR11
  • Method for immunologically measuring soluble LR11

Examples

Experimental program
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preparation example Construction

[0038] For the preparation method of monoclonal antibody using peptide antigen and the method of preparing monoclonal antibody by DNA immunization, refer to the method described in Patent Document 3, and this method is preferably used. Examples of these anti-soluble LR11 monoclonal antibodies include A2-2-3 antibodies obtained by immunizing mice with the synthetic peptide described in Patent Document 3, and mouse monoclonal antibodies obtained by DNA immunization. M3 and M5, rat monoclonal antibodies R14 and R23 obtained by DNA immunization.

[0039] In the measurement method of the present invention, an immunological measurement is performed using a sample treated with one or more of sultaine-type and amide-sultaine-type amphoteric surfactants. This treatment can avoid the influence of substances that interfere with the measurement of soluble LR11 in the sample. Furthermore, even if the reaction time of one reaction is shortened to, for example, 1 hour at room temperature, t...

Embodiment 1

[0072] Example 1 (confirmation of the effect of the amphoteric surfactant of the present invention (sandwich ELISA))

[0073] (1) Preparation of microplates for sandwich ELISA

[0074] In a 96-well microplate (manufactured by NUNC), add 100 μL to each well and dilute to 10 μg with 20 mmol / L phosphate buffer (hereinafter referred to as “PBS”) (pH7.2) containing 150 mmol / L sodium chloride / ml of anti-soluble LR11 monoclonal antibody (M3 antibody), let stand at room temperature for 2 hours to immobilize the M3 antibody. After washing the 96-well microplate with 400 μL of PBS containing 0.05% Tween 20 (registered trademark) (hereinafter referred to as “PBST”), 200 μL of 10% sucrose, PBST containing 1% BSA (hereinafter referred to as for "BSA-PBST"), blocked at room temperature for 2 hours. After removing the liquid in the hole, dry it overnight in a desiccator, seal it in an aluminum bag together with a desiccant, and keep it refrigerated until use.

[0075] (2) Soluble LR11 as...

Embodiment ）

[0081]SB10: catalog number D4266, produced by SIGMA-ALDRICH

[0082] SB12: catalog number A1460, manufactured by Applichem

[0083] SB14: catalog number A1162, manufactured by Applichem

[0084] CHAPS: catalog number C008, produced by Tongren Chemical Research Institute

[0085] CHAPSO: catalog number C020, produced by Tongren Chemical Research Institute

[0086] After washing each well of the microplate three times with 400 μL of PBST, add 100 μL of biotin-labeled anti-soluble LR11 monoclonal antibody (R14 antibody, with biotinylated reagent) diluted to 0.4 μg / mL with BSA-PBST to each well. (manufactured by PIERCE company) for biotin labeling), let stand at room temperature for 4 hours (hereinafter, the biotin-labeled anti-soluble LR11 monoclonal antibody (R14 antibody) at this time and the soluble LR11 captured on the microwell plate in the first reaction The reaction between them is called "secondary reaction"). After the microplate was washed with PBST, 100 μL of perox...

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Abstract

Provided is a method for measuring soluble LR11 in a biospecimen such as blood serum, without requiring isolation of the soluble LR11 therefrom, by using a simple and accurate immunological means. A method for immunologically measuring soluble LR11 in a biospecimen, the method for immunologically measuring the soluble LR11 being characterized in that the specimen is treated using at least one type of surfactant selected from a sulfobetaine amphoteric surfactant and an amidosulfobetaine amphoteric surfactant.

Description

technical field [0001] The present invention relates to a method for the immunological determination of soluble LR11 in a sample of biological origin. Background technique [0002] LDL receptor relative with 11 ligand-binding repeats (LR11) is an LDL receptor-like protein with a molecular weight of about 250 kD that has a characteristic structure in the LDL receptor family (Patent Document 1, Non-Patent Document 1). LR11 (Non-Patent Document 4). LR11 is hardly found in normal blood vessel wall cells, but it has been reported that it can be specifically found in smooth muscle cells of hypertrophic intima (Non-Patent Document 2). In addition, it was reported that with the proliferation of cultured smooth muscle cells, the expression of LR11 was increased, and soluble LR11 was secreted in the culture medium; in the rotator cuff disorder model mice, if the LR11 gene development engineering function was deleted, the migration of smooth muscle cells The hypertrophy of the vascul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/92G01N33/54393G01N33/48G01N33/50G01N33/53
Inventor 田久保耕平海老沼宏幸深町勇武城英明斋藤康
Owner SEKISUI MEDICAL CO LTD
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